Effective pre-selection of sex has been accomplished in many species following the development of reliable methods for separating sperm into enriched X-chromosome and/or Y-chromosome bearing subpopulations. Traditional sperm sorting methods often rely on FACS for the detection of quantifiable differences in DNA content of X-chromosome bearing sperm and Y-chromosome bearing sperm, such as through flow cytometry. A typical step in these methods includes staining a sperm population with a DNA selective fluorescent dye that uniformly stains nuclear DNA. Hoechst 33342, sometimes referred to as Hoechst bisbenzimide 33342, is the most widely utilized stain for this purpose because it can be used in a sufficient quantity to differentiate small variations in nuclear DNA without exhibiting the toxicity of other fluorescent stains.
Sorted sperm quality and purities improved with the introduction of a dead quenching dye for differentiating membrane compromised sperm from the population or populations of interest. Membrane compromised sperm can include dead and dying sperm, which may have fragmented and disintegrating nuclear DNA that obscures the already narrow differences in X-chromosome bearing sperm and Y-chromosome bearing sperm. Initially, propidium iodine was used for this purpose, but has been shown to be toxic to sperm. Later, FD&C red food dye No. 40, 6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfophenyl)azo]-2-naphthalenesulfonic acid (hereafter “red food dye No. 40” or “red 40”), was added to the sperm staining process as the dead quenching dye for quenching membrane compromised sperm and removing membrane compromised sperm from the sort analysis.
When sex sorting sperm, ultimately a small variation in DNA content is quantified differentiating X-chromosome bearing sperm from Y-chromosome bearing sperm. In bovine, for example, Holsteins have about a 3.8% difference in DNA content, while Jersey bulls have about a 4.1% difference. Due to the inexact nature of stoichiometric DNA staining, these small differences can be difficult to differentiate. This difficulty may be exacerbated by noise in the DNA staining complex, which can vary randomly in both X-chromosome bearing sperm and Y-chromosome bearing sperm.
One previously unrealized problem that may exist in current dead quenching staining regiments may be an over quenching of some desired signal. This may occur with the presence of a dead quenching dye in a sperm sample which does not associate with membrane compromised sperm, but instead remains in the sample quenching signals produced by membrane compromised sperm and non-membrane compromised sperm alike. It may also be that some dead quenching dye associates with healthy sperm cells. For these reasons, quenching in the sample may slightly quench the overall signal. While this may be happening to a far extent as compared to the quenching of membrane compromised sperm, it can be relevant when attempting to differentiate small differences in nuclear DNA content.
The sperm sorting process is damaging to cells which are non-regenerative time critical cells. The staining step can be the especially harmful. While Hoechst 33342 can be used in non-toxic concentrations, sperm must be incubated at elevated temperatures and elevated pHs for sufficient Hoechst 33342 penetration with sufficient uniformity for analysis or sorting. Each of elevating sperm temperature and elevating sperm pH may contribute to sperm damage. Therefore, a need exists for improvements to the staining process which reduce stain times or reduce incubation temperatures and/or pHs. Additionally, many chemicals added during sperm processing negatively impact sperm viability. This can be particularly true in sperm staining and sorting procedures.